This year we continued to identify novel antibodies against HIV-1 from our previously constructed and from newly constructed libraries from patients with HIV-1 infection. The major accomplishments are summarized below. 1) We have previously shown that small-size engineered antibodies can target highly conserved epitopes not accessible for full-size antibodies. However, their potency has not been evaluated comparatively with known HIV-1-neutralizing hmAbs and with large panels of primary isolates. We evaluated the inhibitory activity of a single chain antibody fragment (scFv), m9, against several panels of primary HIV-1 isolates from groups M (clades A-G) and N in comparison with the best characterized HIV-1 neutralizing human monoclonal antibodies (hmAbs) using cell-free and cell-associated virus in cell line-based assays. M9 was much more potent than scFv 17b and more potent than or comparable to the best characterized hmAbs IgG1 b12, 2G12, 2F5 and 4E10. It also inhibited cell-to-cell transmission of HIV-1 with higher potency than Fuzeon. We did not find escape mutants resistant to m9 in an vitro assay. These results suggest that m9 is a potential novel anti-HIV-1 candidate therapeutic/prophylactic and its epitope is a new target for small molecules and antibody-based therapeutics. We have also continued to characterize and improve our previously identified potent broadly neutralizing domain antibody (bndAb), m36, which is being tested as potential therapeutics by a major pharmaceutical company. 2) We have proposed that isolated immunoglobulin CH2 domains can be used as scaffolds for selection of binders with potential effector functions (nAbs). We tested the feasibility of this approach by constructing a large (size 5x1010) library where all amino acids in two loops (BC and FG) were mutated to four residues (Y, A, D or S). Three binders were selected from this library by panning against a gp120-CD4 complex. The strongest binder, m1a1, recognized specifically a highly conserved CD4i epitope and inhibited to various extents 7 out of 9 HIV-1 isolates from different clades. These results provide a proof of concept for the potential of CH2 as a scaffold for construction of libraries containing potentially useful binders. The newly identified HIV-1 inhibitors could be further improved to candidate therapeutics and/or used as research reagents for exploration of conserved gp120 structures. 3) Immunological characterization of acute/early HIV-1 infection is important for the design of effective vaccine capable of neutralizing early transmitted virus. However, while recently there is significant progress in our understanding of the nature of such virus, our knowledge of the antibodies elicited during this period and their dynamics is scarce. In particular, there are no such well characterized human mAbs. We constructed phage-displayed antibody libraries from the bone marrow and peripheral blood obtained from an acutely infected patient 40 days and 8 months post infection. Several high-affinity mAbs were selected from these libraries by using recombinant soluble gp120-gp41s (gp140s, Envs) as antigens for panning and screening. Two of these antibodies were more extensively characterized. They bound to the selecting antigen but not to heterologous Envs and had limited neutralizing activity. Compared to the closest germline antibodies they had only several clonally related mutations much less than for known bnAbs, e.g. b12. More extensive characterization of these and newly identified mAbs as well as identification of new mAbs is in progress. These results indicate that during an acute infection the Env could elicit antibodies that are not significantly divergent from the corresponding germline antibodies but of significantly high neutralizing activity to possibly affect the virus dynamics. Further characterization of these antibodies, their dynamics and epitopes could provide knowledge that in addition to its usefulness for basic understanding of immune responses to HIV-1 could also help in the design of candidate vaccine immunogens that elicit potent neutralizers of early transmitted virus. 4) We hypothesized that prolonged exposure to candidate vaccine immunogens could enhance the elicitation of bnAbs. In an attempt to develop candidate HIV-1 vaccine immunogens with prolonged half-lives and increased stability, we constructed a fusion protein, gp41Fc, in which a truncated HIV-1 gp41(89.6) was fused to a human IgG(1) Fc. Gp41Fc is stable in solution, retains its antigenic structure and is highly immunogenic in rabbits. The serum titers reached 1:102,400 for the gp41Fc and 1:5,120 for gp140(89.6). Rabbit IgG neutralized diverse HIV-1 isolates and HIV-2, and the neutralization activity was attributed to gp41-specific IgG. The concentration of the gp41Fc in the serum correlated with the neutralization activity of rabbit IgG which recognized mostly conformation-independent epitopes on gp41 and predominantly bound to peptides derived from the gp41 immunodominant loop region. These results suggest that the prolonged half-life of gp41Fc in the serum may enhance the generation of cross-reactive neutralizing antibodies and may have implications for the development of vaccine immunogens with enhanced capability to elicit cross-reactive HIV-1-neutralizing antibodies. 5) We proposed a novel multi-immunogen approach for elicitation of known broadly neutralizing antibodies by guiding the immune system through complex maturation pathways. It is known that several hmAbs including Fab X5, b12, 2G12 and 2F5 exhibit relatively potent and broad HIV-1 neutralizing activity. These antibodies are highly divergent from the closest corresponding germline antibodies. We have hypothesized that the high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env) to their corresponding germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens. Such immunogens may not be based on the Env but help guide the immune system through the long complex pathways to maturation. We have designed germline-like antibodies where several different CDR3s of the heavy chain were used including one which is identical to that of the mature antibody. The germline-like X5 bound with high affinity to all tested Envs and neutralized a panel of HIV-1 isolates from different clades. In contrast, none of the other germline-like antibodies (b12, 2G12 and 2F5) including antibodies in multivalent formats bound to any of the Envs and did not neutralize any of the HIV-1 isolate tested although the corresponding mature antibodies did with high level of activity. Because b12 is best characterized in terms of structure/function relationships we selected it for characterization of possible maturation pathways. We identified a panel of possible b12 intermediate antibodies and several antigens not related to the Env that bind to some of these antibodies. These antigens together with Envs could be used as a conceptually novel type of candidate vaccines based on two or more immunogens that help guiding the immune system through the complex maturation pathways for elicitation of antibodies that are similar or identical to antibodies with known properties. 6) We initiated a high-throughput sequencing of large portions of the antibody repertoires of acutely infected HIV patients (the HIV antibodyome). We believe that knowledge of the antibodyome will have i [summary truncated at 7800 characters]